Heptahelical terpsichory. Who calls the tune?

The discovery that arrestins can function as ligand-regulated signaling scaffolds has revealed a previously unappreciated level of complexity in G protein-coupled receptor (GPCR) signal transduction. Because arrestin-bound GPCRs are uncoupled from G proteins, arrestin binding can be viewed as switching receptors between two temporally and spatially distinct signaling modes. Recent work has established two factors that underscore this duality of GPCR signaling and suggest it may ultimately have therapeutic significance. The first is that signaling by receptor-arrestin "signalsomes" does not require heterotrimeric G protein activation. The second is that arrestin-dependent signals can be initiated by pathway-specific "biased agonists," creating the potential for drugs that selectively modulate different aspects of GPCR function. Currently, however, little is known about the physiological relevance of G protein-independent signals at the cellular or whole animal levels, and additional work is needed to determine whether arrestin pathway-selective drugs will find clinical application.     

Arrestin interaction with rhodopsin

It is becoming increasingly apparent that G protein-coupled receptors (GPCRs) can exist and function as oligomers. This notion differs from the classical view of signaling wherein the receptor has been presumed to be monomeric. Despite this shift in views, the interpretation of data related to GPCR function is still largely carried out within the framework of a monomeric receptor. Rhodopsin is a prototypical GPCR that initiates phototransduction. Like other GPCRs, the activity of rhodopsin is regulated by phosphorylation and the binding of arrestin. In the current investigation, we have explored by modeling methods the interaction of rhodopsin and arrestin under the assumption that either one or two rhodopsin molecules bind each arrestin molecule. The dimeric receptor framework may provide a more accurate representation of the system and is therefore likely to lead to a better and more accurate understanding of GPCR signaling.     

Structural biology of G protein‐coupled receptor signaling complexes

G protein‐coupled receptors (GPCRs) constitute the largest family of cell surface receptors that mediate numerous cell signaling pathways, and are targets of more than one‐third of clinical drugs. Thanks to the advancement of novel structural biology technologies, high‐resolution structures of GPCRs in complex with their signaling transducers, including G‐protein and arrestin, have been determined. These 3D complex structures have significantly improved our understanding of the molecular mechanism of GPCR signaling and provided a structural basis for signaling‐biased drug discovery targeting GPCRs. Here we summarize structural studies of GPCR signaling complexes with G protein and arrestin using rhodopsin as a model system, and highlight the key features of GPCR conformational states in biased signaling including the sequence motifs of receptor TM6 that determine selective coupling of G proteins, and the phosphorylation codes of GPCRs for arrestin recruitment. We envision the future of GPCR structural biology not only to solve more high‐resolution complex structures but also to show stepwise GPCR signaling complex assembly and disassembly and dynamic process of GPCR signal transduction.     

A Novel Polar Core and Weakly Fixed C-Tail in Squid Arrestin Provide New Insight into Interaction with Rhodopsin

Photoreceptors of the squid Loligo pealei contain a G-protein-coupled receptor (GPCR) signaling system that activates phospholipase C in response to light. Analogous to the mammalian visual system, signaling of the photoactivated GPCR rhodopsin is terminated by binding of squid arrestin (sArr). sArr forms a light-dependent, high-affinity complex with squid rhodopsin, which does not require prior receptor phosphorylation for interaction. This is at odds with classical mammalian GPCR desensitization where an agonist-bound phosphorylated receptor is needed to break stabilizing constraints within arrestins, the so-called “three-element interaction” and “polar core” network, before a stable receptor–arrestin complex can be established. Biophysical and mass spectrometric analysis of the squid rhodopsin–arrestin complex indicates that in contrast to mammalian arrestins, the sArr C-tail is not involved in a stable three-element interaction. We determined the crystal structure of C-terminally truncated sArr that adopts a basal conformation common to arrestins and is stabilized by a series of weak but novel polar core interactions. Unlike mammalian arrestin-1, deletion of the sArr C-tail does not influence kinetic properties of complex formation of sArr with the receptor. Hydrogen–deuterium exchange studies revealed the footprint of the light-activated rhodopsin on sArr. Furthermore, double electron–electron resonance spectroscopy experiments provide evidence that receptor-bound sArr adopts a conformation different from the one known for arrestin-1 and molecular dynamics simulations reveal the residues that account for the weak three-element interaction. Insights gleaned from studying this system add to our general understanding of GPCR–arrestin interaction.     

Clathrin-dependent mechanisms of G protein-coupled receptor endocytosis

The heptahelical G protein-coupled receptor (GPCR) family includes approximately 900 members and is the largest family of signaling receptors encoded in the mammalian genome. G protein-coupled receptors elicit cellular responses to diverse extracellular stimuli at the plasma membrane and some internalized receptors continue to signal from intracellular compartments. In addition to rapid desensitization, receptor trafficking is critical for regulation of the temporal and spatial aspects of GPCR signaling. Indeed, GPCR internalization functions to control signal termination and propagation as well as receptor resensitization. Our knowledge of the mechanisms that regulate mammalian GPCR endocytosis is based predominantly on arrestin regulation of receptors through a clathrin- and dynamin-dependent pathway. However, multiple clathrin adaptors, which recognize distinct endocytic signals, are now known to function in clathrin-mediated endocytosis of diverse cargo. Given the vast number and diversity of GPCRs, the complexity of clathrin-mediated endocytosis and the discovery of multiple clathrin adaptors, a single universal mechanism controlling endocytosis of all mammalian GPCRs is unlikely. Indeed, several recent studies now suggest that endocytosis of different GPCRs is regulated by distinct mechanisms and clathrin adaptors. In this review, we discuss the diverse mechanisms that regulate clathrin-dependent GPCR endocytosis.     

Ascidian arrestin (Ci-arr), the origin of the visual and nonvisual arrestins of vertebrate.

Arrestin is one of the key proteins for the termination of G protein signaling. Activated G protein-coupled receptors (GPCRs) are specifically phosphorylated by G protein-coupled receptor kinases (GRKs) and then bind to arrestins to preclude the receptor/G protein interaction, resulting in quenching of the following signal transduction. Vertebrates possess two types of arrestin; visual arrestin expressed exclusively in photoreceptor cells in retinae and pineal organs, and beta-arrestin, which is expressed ubiquitously. Unlike visual arrestin, beta-arrestin contains the clathrin-binding domain at the C-terminus, responsible for the agonist-induced internalization of GPCRs. Here, we isolated a novel arrestin gene (Ci-arr) from the primitive chordate, the ascidian Ciona intestinalis larvae. The deduced amino acid sequence suggests that Ci-Arr be closely related to vertebrate arrestins. Interestingly, this arrestin has the feature of both visual and beta-arrestin. Whereas the expression of Ci-arr was restricted to the photoreceptors in the larvae similarly to visual arrestin, the gene product, containing the clathrin-binding domain, promoted the GPCR internalization in HEK293tsA201 cells similarly to beta-arrestin. The phylogenetic tree shows that Ci-Arr is branched from a common root of visual and beta-arrestins. Southern analysis suggests that the Ciona genome contains only one gene for the arrestin family. These results suggest that the visual and beta-arrestin genes were generated by the duplication of the prototypical arrestin gene like Ci-arr in the early evolution of vertebrates.     

Desensitization of herpesvirus-encoded G protein-coupled receptors

Members of the herpesvirus family, including human cytomegalovirus (HCMV) and Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8), encode G protein-coupled receptor (GPCR) homologs, which strongly activate classical G protein signal transduction networks within the cell. In animal models of herpesvirus infection, the viral GPCRs appear to play physiologically important roles by enabling viral replication within tropic tissues and by promoting reactivation from latency. While a number of studies have defined intracellular signaling pathways activated by herpesviral GPCRs, it remains unclear if their physiological function is subjected to the process of desensitization as observed for cellular GPCRs. G protein-coupled receptor kinases (GRK) and arrestin proteins have been recently implicated in regulating viral GPCR signaling; however, the role that these desensitization proteins play in viral GPCR function in vivo remains unknown. Here, we review what is currently known regarding viral GPCR desensitization and discuss potential biological ramifications of viral GPCR regulation by the host cell desensitization machinery.     

Structural Features of β2 Adrenergic Receptor: Crystal Structures and Beyond

The beta2-adrenergic receptor (β2AR) family, which is the largest family of cell surface receptors in humans. Extra attention has been focused on the human GPCRs because they have been studied as important protein targets for pharmaceutical drug development. In fact, approximately 40% of marketed drugs directly work on GPCRs. GPCRs respond to various extracellular stimuli, such as sensory signals, neurotransmitters, chemokines, and hormones, to induce structural changes at the cytoplasmic surface, activating downstream signaling pathways, primarily through interactions with heterotrimeric G proteins or through G-protein independent pathways, such as arrestin. Most GPCRs, except for rhodhopsin, which contains covalently linked 11 cis-retinal, bind to diffusible ligands, having various conformational states between inactive and active structures. The first human GPCR structure was determined using an inverse agonist bound β2AR in 2007 and since then, more than 20 distinct GPCR structures have been solved. However, most GPCR structures were solved as inactive forms, and an agonist bound fully active structure is still hard to obtain. In a structural point of view, β2AR is relatively well studied since its fully active structure as a complex with G protein as well as several inactive structures are available. The structural comparison of inactive and active states gives an important clue in understanding the activation mechanism of β2AR. In this review, structural features of inactive and active states of β2AR, the interaction of β2AR with heterotrimeric G protein, and the comparison with β1AR will be discussed.     

Conservation of the phosphate-sensitive elements in the arrestin family of proteins.

Arrestins play a key role in the homologous desensitization of G protein-coupled receptors (GPCRs). These cytosolic proteins selectively bind to the agonist-activated and GPCR kinase-phosphorylated forms of the GPCR, precluding its further interaction with the G protein. Certain mutations in visual arrestin yield "constitutively active" proteins that bind with high affinity to the light-activated form of rhodopsin without requiring phosphorylation. The crystal structure of visual arrestin shows that these activating mutations perturb two groups of intramolecular interactions that keep arrestin in its basal (inactive) state. Here we introduced homologous mutations into arrestin2 and arrestin3 and found that the resulting mutants bind to the beta(2)-adrenoreceptor in vitro in a phosphorylation-independent fashion. The same mutants effectively desensitize both the beta(2)-adrenergic and delta-opioid receptors in the absence of receptor phosphorylation in Xenopus oocytes. Moreover, the arrestin mutants also desensitize the truncated delta-opioid receptor from which the C terminus, containing critical phosphorylation sites, has been removed. Conservation of the phosphate-sensitive hot spots in non-visual arrestins suggests that the overall fold is similar to that of visual arrestin and that the mechanisms whereby receptor-attached phosphates drive arrestin transition into the active binding competent state are conserved throughout the arrestin family of proteins.     

N-formyl peptide receptor phosphorylation domains differentially regulate arrestin and agonist affinity

Arrestins regulate the signaling and endocytosis of many G protein-coupled receptors (GPCRs). It has been suggested that the functions of arrestins are dependent upon both the number and pattern of phosphorylation sites present in an activated GPCR. However, little is currently known about the relationships between the sites of receptor phosphorylation, the resulting affinities of arrestin binding, and the ensuing mechanisms of receptor regulation for any given GPCR. To investigate these interactions, we used an active truncated mutant of arrestin (amino acids 1-382) and phosphorylation-deficient mutants of the N-formyl peptide receptor (FPR). In contrast to results with wild type arrestins, the truncated arrestin-2 protein bound to the unphosphorylated wild type FPR, although with lower affinity and a low affinity for the agonist as revealed by competition studies with heterotrimeric G proteins. Using FPR mutants, we further demonstrated that the phosphorylation status of serines and threonines between residues 328-332 is a key determinant that regulates the affinity of the FPR for arrestins. Furthermore, we found that the phosphorylation status of serine and threonine residues between amino acids 334 and 339 regulates the affinity of the receptor for agonist when arrestin is bound. These results suggest that the agonist affinity state of the receptor is principally regulated by phosphorylation at specific sites and is not simply a consequence of arrestin binding as has previously been proposed. Furthermore, this is the first demonstration that agonist affinity of a GPCR and the affinity of arrestin binding to the phosphorylated receptor are regulated by distinct receptor phosphodomains.     

A beta-arrestin binding determinant common to the second intracellular loops of rhodopsin family G protein-coupled receptors

beta-Arrestins have been shown to inhibit competitively G protein-dependent signaling and to mediate endocytosis for many of the hundreds of nonvisual rhodopsin family G protein-coupled receptors (GPCR). An open question of fundamental importance concerning the regulation of signal transduction of several hundred rhodopsin-like GPCRs is how these receptors of limited sequence homology, when considered in toto, can all recruit and activate the two highly conserved beta-arrestin proteins as part of their signaling/desensitization process. Although the serine and threonine residues that form GPCR kinase phosphorylation sites are common beta-arrestin-associated receptor determinants regulating receptor desensitization and internalization, the agonist-activated conformation of a GPCR probably reveals the most fundamental determinant mediating the GPCR and arrestin interaction. Here we identified a beta-arrestin binding determinant common to the rhodopsin family GPCRs formed from the proximal 10 residues of the second intracellular loop. We demonstrated by both gain and loss of function studies for the serotonin 2C, beta2-adrenergic, alpha2a)adrenergic, and neuropeptide Y type 2 receptors that the highly conserved amino acids, proline and alanine, naturally occurring in rhodopsin family receptors six residues distal to the highly conserved second loop DRY motif regulate beta-arrestin binding and beta-arrestin-mediated internalization. In particular, as demonstrated for the beta2 AR, this occurs independently of changes in GPCR kinase phosphorylation. These results suggest that a GPCR conformation directed by the second intracellular loop, likely using the loop itself as a binding patch, may function as a switch for transitioning beta-arrestin from its inactive form to its active receptor-binding state.     

Decoding the signaling of a GPCR heteromeric complex reveals a unifying mechanism of action of antipsychotic drugs

Atypical antipsychotic drugs, such as clozapine and risperidone, have a high affinity for the serotonin 5-HT(2A) G protein-coupled receptor (GPCR), the 2AR, which signals via a G(q) heterotrimeric G protein. The closely related non-antipsychotic drugs, such as ritanserin and methysergide, also block 2AR function, but they lack comparable neuropsychological effects. Why some but not all 2AR inhibitors exhibit antipsychotic properties remains unresolved. We now show that a heteromeric complex between the?2AR and the G(i)-linked GPCR, metabotropic glutamate 2 receptor (mGluR2), integrates ligand input,?modulating signaling output and behavioral changes. Serotonergic and glutamatergic drugs bind the mGluR2/2AR heterocomplex, which then balances Gi- and Gq-dependent signaling. We find that the mGluR2/2AR-mediated changes in Gi and Gq activity predict the psychoactive behavioral effects of a variety of pharmocological compounds. These observations provide mechanistic insight into antipsychotic action that may advance therapeutic strategies for disorders including schizophrenia and dementia.     

G protein-coupled receptor kinase function is essential for chemosensation in C. elegans

G protein-coupled receptors (GPCRs) mediate diverse signaling processes, including olfaction. G protein-coupled receptor kinases (GRKs) are important regulators of G protein signal transduction that specifically phosphorylate activated GPCRs to terminate signaling. Despite previously described roles for GRKs in GPCR signal downregulation, animals lacking C. elegans G protein-coupled receptor kinase-2 (Ce-grk-2) function are not hypersensitive to odorants. Instead, decreased Ce-grk-2 function in adult sensory neurons profoundly disrupts chemosensation, based on both behavioral analysis and Ca(2+) imaging. Although mammalian arrestin proteins cooperate with GRKs in receptor desensitization, loss of C. elegans arrestin-1 (arr-1) does not disrupt chemosensation. Either overexpression of the C. elegans Galpha subunit odr-3 or loss of eat-16, which encodes a regulator of G protein signaling (RGS) protein, restores chemosensation in Ce-grk-2 mutants. These results demonstrate that loss of GRK function can lead to reduced GPCR signal transduction and suggest an important role for RGS proteins in the regulation of chemosensation.     

Regulators of G protein signaling (RGS proteins): novel central nervous system drug targets.

Many drugs of abuse signal through receptors that couple to G proteins (GPCRs), so the factors that control GPCR signaling are likely to be important to the understanding of drug abuse. Contributions by the recently identified protein family, regulators of G protein signaling (RGS) to the control of GPCR function are just beginning to be understood. RGS proteins can accelerate the deactivation of G proteins by 1000-fold and in cell systems they profoundly inhibit signaling by many receptors, including mu-opioid receptors. Coupled with the known dynamic regulation of RGS protein expression and function, they are of obvious interest in understanding tolerance and dependence mechanisms. Furthermore, drugs that could inhibit their activity could be useful in preventing the development of or in treating drug dependence.     

Phosphorylation modulates the affinity of light-activated rhodopsin for G protein and arrestin

Reduced effector activity and binding of arrestin are widely accepted consequences of GPCR phosphorylation. However, the effect of receptor multiphosphorylation on G protein activation and arrestin binding parameters has not previously been quantitatively examined. We have found receptor phosphorylation to alter both G protein and arrestin binding constants for light-activated rhodopsin in proportion to phosphorylation stoichiometry. Rod disk membranes containing different average receptor phosphorylation stoichiometries were combined with G protein or arrestin, and titrated with a series of brief light flashes. Binding of G(t) or arrestin to activated rhodopsin augmented the 390 nm MII optical absorption signal by stabilizing MII as MII.G or MII.Arr. The concentration of active arrestin or G(t) and the binding constant of each to MII were determined using a nonlinear least-squares (Simplex) reaction model analysis of the titration data. The binding affinity of phosphorylated MII for G(t) decreased while that for arrestin increased with each added phosphate. G(t) binds more tightly to MII at phosphorylation levels less than or equal to two phosphates per rhodopsin; at higher phosphorylation levels, arrestin binding is favored. However, arrestin was found to bind much more slowly than G(t) at all phosphorylation levels, perhaps allowing time for phosphorylation to gradually reduce receptor-G protein interaction before arrestin capping of rhodopsin. Sensitivity of the binding constants to ionic strength suggests that a strong membrane electrostatic component is involved in both the reduction of G(t) binding and the increase of arrestin binding with increasing rhodopsin phosphorylation.     

Novel roles for arrestins in the post-endocytic trafficking of G protein-coupled receptors

G protein-coupled receptors (GPCRs) represent the largest family of transmembrane signaling molecules in the human genome. As such, they interact with numerous intracellular molecules, which can act either to propagate or curtail signaling from the receptor. Their primary mode of cellular activation occurs through heterotrimeric G proteins, which in turn can activate a wide spectrum of effector molecules, including phosphodiesterases, phospholipases, adenylyl cyclases and ion channels. Active GPCRs are also the target of G protein-coupled receptor kinases, which phosphorylate the receptors culminating in the binding of the protein arrestin. This results in rapid desensitization through inhibition of G protein binding, as well as novel mechanisms of cellular activation that involve the scaffolding of cellular kinases to GPCR-arrestin complexes. Arrestins can also serve to mediate the internalization of certain GPCRs, a process which plays an important role in regulating cellular activity both by mediating long-term desensitization through down regulation (degradation) of receptors and by recycling desensitized receptors back to the cell surface to initiate additional rounds of signaling. The mechanisms that regulate the subsequent intracellular trafficking of GPCRs following internalization are largely unknown. Recently however, it has become clear that the pattern of receptor phosphorylation and subsequent binding of arrestin play a critical role in the intracellular trafficking of internalized receptors, thereby dictating the ultimate fate of the receptor. In addition, arrestins have now been shown to be required for the recycling of GPCRs that are capable of internalizing through arrestin-independent mechanisms. This review will summarize recent advances in our understanding of the roles of arrestins in post-endocytic GPCR trafficking.     

Mouse cones require an arrestin for normal inactivation of phototransduction

Arrestins are proteins that arrest the activity of G protein-coupled receptors (GPCRs). While it is well established that normal inactivation of photoexcited rhodopsin, the GPCR of rod phototransduction, requires arrestin (Arr1), it has been controversial whether the same requirement holds for cone opsin inactivation. Mouse cone photoreceptors express two distinct visual arrestins: Arr1 and Arr4. By means of recordings from cones of mice with one or both arrestins knocked out, this investigation establishes that a visual arrestin is required for normal cone inactivation. Arrestin-independent inactivation is 70-fold more rapid in cones than in rods, however. Dual arrestin expression in cones could be a holdover from ancient genome duplication events that led to multiple isoforms of arrestin, allowing evolutionary specialization of one form while the other maintains the basic function.     

Modulation of methuselah expression targeted to Drosophila insulin‐producing cells extends life and enhances oxidative stress resistance

Ubiquitously reduced signaling via Methuselah (MTH), a G‐protein‐coupled receptor (GPCR) required for neurosecretion, has previously been reported to extend life and enhance stress resistance in flies. Whether these effects are due to reduced MTH signalling in specific tissues remains unknown. We determined that reduced expression of mth targeted to the insulin‐producing cells (IPCs) of the fly brain was sufficient to extend life and enhance oxidative stress resistance. Paradoxically, we discovered that overexpression of mth targeted to the same cells has similar phenotypic effects to reduced expression due to MTH's interaction with β‐arrestin, which uncouples GPCRs from their G‐proteins. We confirmed the functional relationship between MTH and β‐arrestin by finding that IPC‐targeted overexpression of β‐arrestin alone mimics the longevity phenotype of reduced MTH signaling. As reduced MTH signaling also inhibits insulin secretion from the IPCs, the most parsimonious mechanistic explanation of its longevity and stress‐resistance enhancement might be through reduced insulin/IGF signaling (IIS). However, examination of phenotypic features of long‐lived IPC‐mth modulated flies as well as several downstream IIS targets implicates enhanced activity of the JNK stress‐resistance pathway more directly than insulin signaling in the longevity and stress‐resistance phenotypes.     

Heterotrimeric G proteins demonstrate differential sensitivity to β-arrestin dependent desensitization

G15 is a heterotrimeric G protein of the Gq/11 family. In this study, we describe its exceptional poor sensitivity to the general regulatory mechanism of G protein-coupled receptor (GPCR) desensitization.Enhancing β2 adrenergic receptor desensitization by arrestin overexpression, did not affect signalling to G15. Similarly, increased levels of arrestin did not affect G15 signalling triggered by the activation of V2 vasopressin and δ opioid receptors.Furthermore, co-immunoprecipitation experiments showed that G15 α subunit (as opposed to Gαq and Gαs) is recruited to a V2 vasopressin receptor mutant that is constitutively desensitized by β-arrestin. Interestingly, co-expression of Gα15 partially rescued cell surface localization and signalling capabilities of the same mutant receptor and reduced β2 adrenergic receptor internalization.Taken together, these findings provide evidence for a novel mechanism whereby GPCR desensitization can be bypassed and G15 can support sustained signalling in cells chronically exposed to hormones or neurotransmitters.     

Adrenal angiotensin II type 1 receptor biased signaling: The case for “biased” inverse agonism for effective aldosterone suppression

Angiotensin II (AngII) uses two distinct G protein-coupled receptor (GPCR) types, AT₁R and AT₂R, to exert a plethora of physiologic effects in the body and to significantly affect cardiovascular homeostasis. Although not much is known about the signaling of the AT₂R, AT₁R signaling is known to be quite pleiotropic, mobilizing a variety of signal transducers inside cells to produce a biological outcome. When the outcome in question is aldosterone production from the adrenal cortex, the main transducers activated specifically by the adrenocortical AT₁R to signal toward that cellular effect are the G_q/11 protein alpha subunits and the β-arrestins (also known as Arrestin-2 and -3). The existence of various downstream pathways the AT₁R signal can travel down on has led to the ever-expanding filed of GPCR pharmacology termed “biased” signaling, which refers to a ligand preferentially activating one signaling pathway over others downstream of the same receptor in the same cell. However, “biased” signaling or “biased” agonism is therapeutically desirable only when the downstream pathways lead to different or opposite cellular outcomes, so the pathway promoting the beneficial effect can be selectively activated over the pathway that leads to detrimental consequences. In the case of the adrenal AT₁R, both G_q/11 proteins and β-arrestins mediate signaling to the same end-result: aldosterone synthesis and secretion. Therefore, both pathways need to remain inactive in the adrenal cortex to fully suppress the production of aldosterone, which is one of the culprit hormones elevated in chronic heart failure, hypertension, and various other cardiovascular diseases. Variations in the effectiveness of the AT₁R antagonists, which constitute the angiotensin receptor blocker (ARB) class of drugs (also known as sartans), at the relative blockade of these two pathways downstream of the adrenal AT₁R opens the door to the flip term “biased” inverse agonism at the AT₁R. ARBs that are unbiased and equipotent inverse agonists for both G proteins and β-arrestins at this receptor, like candesartan and valsartan, are the most preferred agents with the best efficacy at reducing circulating aldosterone, thereby ameliorating heart failure. In the present review, the biased signaling of the adrenal AT₁R, particularly in relation to aldosterone production, is examined and the term “biased” inverse agonism at the AT₁R is introduced and explained, as a means of pharmacological categorization of the various agents within the ARB drug class.